This invention relates to a pharmaceutical formulation useful for the treatment of Hepatitis B and Hepatitis C and other viral infections of liver. This invention particularly relates to a pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B and C virus infections prepared from the Indian biotyped medicinal plant, Phyllanthus amarus. This invention also relates to a process for the preparation of the pharmaceutical formulation useful for the treatment of acute and chronic Hepatitis B and Hepatitis C and other viral infections of the liver from the medicinal plant Phyllanthus amarus. 
It is needless to stress the need for a s drug that would keep the liver functioning at its optimum or the one that would be selectively active against the currently known etiological agents of acute and chronic viral diseases of the liver. This is important because the disease of the liver throw the entire human body out of gear. The exciting alphabet of viral Hepatitis includes a wide range of totally unrelated often highly unusual pathogenic human viruses like Hepatitis A virus (HAV), Hepatitis B virus (HSV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatits E virus (HEV) etc. Of the viruses it has been clearly established that HBV, HCV and HDV are the ones that are associated with the development of chronic persistent/active hepatitis, cirrhosis of the liver and even hepatocellular carcinoma besides being associated with fulminant hepatitis and sub acute hepatic failure.
Acute and Chronic Hepatitis-B
The natural disease course in HBV is being summarized to understand the need for the effective management and treatment of Hepatitis B in a country. For normal adults with low viral production and an early immune response, the disease course is self limiting and usually asymptomatic (60-80% of all HBV infections). Individuals who replicate the virus in larger quantities, with a relatively late immune response have a self-limiting symptomatic acute hepatitis. Irrespective of whether initially symptomatic or asymptomatic, the infection becomes chronic in 5-10% of the individuals, 20-30% of them developing clinical sequelae such as chronic hepatitis, cirrhosis or hepatoma within years or decades. In neonates, however, the immune defence is still lacking (induction of tolerance), so that infected individuals do not develop acute hepatitis, but more of them become chronic camers (80-90%). Such carriers also progress frequently to chronic clinical sequelae faster. Between these two extremes are immunocompromised individuals, such as intravenous drug users, haemodialysis patients or transplant recipients, who are more likely to become chronic carriers than are healthy adults (10-60%). (WHO Tech. Report Series 1987;754:18).
Based on substantial body of data, HBV has been proved as a major pathogen producing chronic liver diseases. It has also been proved that there are over 400 million healthy carriers of HBV all over the world and one tenth of these carriers (40 millions) being in India alone. These carriers besides acting as human reservoirs of HBV infection also act as primary source of spread of HBV infection to the community and was also shown to have 200 times increased risk of developing chronic liver diseases and/or hepatocellular carcinoma.
With the above documented international HBV scenario, the HBV epidemiology in India is to be considered as alarming since there are definite data on prevalence pattern of HBV in asymptomatic population (4%) high risk groups (13%), significant involvement of HBV in Indian acute and sub-acute liver failure cases (42 and 45%). 70% of the chronic hepatitis cases, 40-80% of cirrhosis cases and over 60% of primary liver cancer cases.
Although effective vaccines have been developed against HBV and successfully adopted, the need for effective treatment of acute and chronic Hepatitis B has become universal public health emergency since vaccines as on date are neither capable of inducing immunity in a carrier nor able to eliminate HBV carrier status. Research conducted from the mid 70s have delineated several agents to have treatment potential in chronic HBV infections which has been illustrated in the Table 1 given below.
However, except the interferons, Lamuvidine and the latest entry Phyllanthus amarus, the others seem to be far from successful. The limited success rate, prohibitive cost, profound side effects and the non-accessibility of interferons and Lamuvidine in developing and underdeveloped counties have necessitated further search for newer antihepatitis B agents.
Acute and Chronic Hepatitis-C
1. Acute HCV Infection
Persons with acute HCV infection typically are either asymptomatic or have a mild clinical illness; 60%-70% have no discernible symptoms; 20%-30% might have jaundice; and 10%-20% might have nonspecific symptoms (e.g. anorexia, malaise or abdominal pain). Clinical illness in patients with acute hepatitis C who seek medical care is similar to that of other types of viral hepatitis, and serologic testing is necessary to determine the etiology of hepatitis in an individual patient. In xe2x89xa720% of these patients, onset of symptoms might precede anti-HCV seroconversion. Average time period from exposure to symptom onset is 6-7 weeks, whereas average time period from exposure to seroconversion is 8-9 weeks. Anti-HCV can be detected in 80% of patients within 15 weeks after exposure, in xe2x89xa790% within 5 months after exposure, and in xe2x89xa797% by 6 months after exposure. Rarely, seroconversion might be delayed until 9 months after exposure.
The course of acute hepatitis C is variable, although elevations in serum ALT levels often in a fluctuating pattern, are its most characteristic feature. Normalization of ALT levels might occur and suggests full recovery, but this is frequently followed by ALT elevations that indicate progression to chronic disease. Fulminant hepatic failure following acute hepatitis C is rare. However, in developing country especially India, HCV in FHF was reported significantly.
2. Chronic HCV Infection
After acute infection, 15%-25% of persons appear to resolve their infection without sequelae as defined by substained absence of HCV RNA in serum and normalization of ALT levels. Chronic HCV infection develops in most persons (75%-85%), with persistent or fluctuating ALT elevations indicating active liver disease developing in 60%-70% of chronically infected persons. In the remaining 30%-40% of chronically infected persons, ALT levels are normal. No clinical or epidemiologic features among patients with acute infection have been found to be predictive of either persistent infection or chronic liver disease. Moreover, various ALT patterns have been observed in these patients during follow-up, and patients might have prolonged periods (xe2x89xa712 months) of normal ALT activity even though they have histologically confirmed chronic hepatitis. Thus a single ALT determination cannot be used to exclude ongoing hepatic injury, and long term follow-up of patients with HCV infection is required to determine their clinical outcome or prognosis.
The course of chronic liver disease is usually insidious, progressing at a slow rate without symptoms or physical signs in the majority of patients during the first two or more decades after infection. Frequently, chronic hepatitis C is not recognised until asymptomatic persons are identified as HCV positive during blood donor screening or elevated ALT levels are detected during routine physical examinations. Most studies have reported that cirrhosis develops in 10%-20% of persons with chronic hepatitis C over a period of 20-30 years, and
HCC in 1%-5%, with striking geographic variations in rates of this disease. This difference is more discernable also in the SEA region countries.
Although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged  greater than 40 years at infection, and being male are associated with more severe liver disease. In particular, among persons with alcoholic liver disease and HCV infection, liver disease progresses more rapidly; among those with cirrhosis, a higher risk for development of HCC exists. Furthermore, even intake of moderate amounts ( greater than 10 g/day) of alcohol in patients with chronic hepatitis C might enhance disease progression. More severe liver injury observed in persons with alcoholic liver disease and HCV infection possibly is attributable to alcohol induced enhancement of viral replication or increased susceptibility of cells to viral injury.
The current treatment guidelines of HCV infection are presented below:
Current effective therapy for HCV infection is IFN based, with or without other therapeutic agents such as ribavirin. Ribavirin monotherapy is not recommended.
For the interim, patients with acute hepatitis should receive IFN-xcex1, 3-6 million units (or 9-15 xcexcg) thrice weekly for at least 6 months until more effective regimens emerge.
The standard treatment for previously untreated (naxc3xafve) patients with chronic hepatitis C is IFN-xcex1, 36 million units (or 915 xcexcg) thrice weekly for 12 months. However, recent data indicate that a regimen of IFN-xcex1 and ribavirin for 6 months or IFN-xcex1 monotherapy using different schedules and/or higher doses may significantly improve sustained response rates and become preferred options for treatment in the future.
Adverse sideeffects to IFN and ribavirin are tolerable, but a fatal outcome (suicide, liver failure, sepsis) has been observed primarily in patients with cirrhosis. Less severe side effects occur in less than 10% of the treated patients and include flu-like symptoms, fatigue, thinning of hair, myalgia, bone marrow suppression requiring dose reduction,
neuropsychiatric effects, such as depression and autoimmune disease (thyroid). All patients must be carefully monitored by the prescribing doctor for side-effects by using appropriate.
biochemical, haematological and immunological tests. Appropriate medical records should be maintained.
Newer Search of Antiviral Agents Against Hepatitis B and Hepatitis C
One of these searches for the last two decades has been in the development of a promising antiviral agent against hepatitis B and hepatitis C and other viral infections of liver from the plant, Phyllanthus amarus. 
Phyllanthus niruri Linn. as it has been indexed in majority of published ethano botanical reviews, until recently, belongs to the family Euphorbiaceae. Phyllanthus is one of the largest genera of the family Euphorbiaceae containing about 700 species. It has been shown that about 24 species of Phyllanthus are active against clinical Hepatits (jaundice) as indicated in Table 2, out of which 8 have been used in India.
Taxonomy of Phyllanthus Amarus 
This plant has recently been delineated as a mixture of three distinct species namely Phyllanthus amarus, Phyllanthus fraternus and Phyllanthus debilis. It was later identified that the cirumtropical weed P. amarus is the predominant species in South India, particularly in Tamilnadu. P. amarus are erect annual herbs, 10-60 cm tall; main stem simple or branched, terrete smooth or scabridulous in younger parts. Cataphylls, stipules 1.5-1.9 mm long, deltoid acuminate blade 1-1.5 mm long, subulate acuminate. Deciducus branchlets 1.5-14 cms long, subserete, smooth or a few lower nodes sometimes scabridulcus with 13-30 distichous leaves. Leaves 3-11xc3x971.5xc3x976 mm elliptic oblong obovate, oblong, or even obovate, obtuse, or minutely apiculate at apex, obtuse or slightly inequilateral at base, petioles 0.3-0.5 mm long, stipules 0.8-1.1 mm long triangular accuminate. Flowers in axillary, unsexual and bisexual cymles on deciduous branches. Proximal 2-3 axis with unisexual cymules, each consisting of 1 male and 1 female or 2(xe2x88x923) males and female or 1 male and 2 female flower or combination thereof; male flowers pedicals at anthesis ca 1 mm long. Calyx lobe 5, subequal each ca 0.7xc3x970.3 mm elliptic or oblong elliptic and abruptly acute at apex hyaline with unbranched mid ribs. Disc segments 5, roundish stames 3 (rarely 2): filaments connate into a column 0.2-0.3 mm high autheros sessile a top dehiscing longitudinally. Female flowers; pedicles 0.8-1 mm long, obtusely 4 gonous, dialated above, ca 1.5 mm in fruits, calyx 5 lobes, subequal. Ovate-oblong, acute at apex, midsepaline band green. Disc flat, deeply 5 lobes. Lobes sometimes toothed at apex. Styles 3, free, more or less spreading, shallowly bifid at apex; arms divergent (Mitra and Jain, Bull Bot Surv Ind 1985;27:167-176).
Historical Use of P. Niruri in Jaundice
Even though clinical uses of P. niruri and other species vz. P. amarus cited for over a century in the Ayurvedha and Siddha literatures, scientific evaluatory studies have been attempted only during the last 50 years for its efficacy in the treatment of jaundice/viral hepatitis. A logical approach towards identification of the active principles of P. amarus is to fractionate the plant extracts and identify biologically active compounds and to chemically characterise them.
Studies on P. Niruri/P. Amarus Against HBV
The first ever designed invitro antiviral study on Phyllanthus niruri against any hepatitis virus with HBV as model was reported by Thyagarajan in 1979 from Madras (Thyagarajan, Ph.D. Thesis, University of Madras 1979), India. Subsequently, Thyagarajan et al (1982) have shown the whole plant extract of P. niruri through several solvents brought about binding of Hepatitis B surface antigen (HBsAg) (Thygarajan et al., Ind J Med Res 1982; 76(Suppl.): 124-130. This plant from Tamilnadu, India was later identified taxonomically by Unander as P. amarus. Venkateswaran et al (1987) (Proc Natl Acad Sci USA 1989;14:195-201) and Blumberg et al (1989) (Cancer detection and prevention 1987;84:274-278). from United States using the P. amarus plants provided by Thyagarajan have shown that the plants collected from Madras, India whose aqueous extracts bound the surface antigen of HBV invitro, have inhibited the viral DNA polymerase (DNAp) of HBV and Woodchuck hepatitis virus (WHV) invitro. When administered intraperitoneally to WHV infected woodchucks, acutely infected animals lost the viral surface antigen; the surface antigen titre dropped in some chronically infected animals; the liver cancer rat in treated chronically infected animals was lower than the untreated controls.
Based on these findings they had secured an Australian patent numbered AV-A-56530/86 for a composition of matter useful in the treatment of Hepatitis B virus infection consisting essentially of the methanol extractable components of P. niruri L.
Yanagi et al., (Meeting on Hepatis viruses, Sep. 25-28, 1989, Cold Spring Haror Laboratory, NY, 1989,77) from Japan have reported at aqueous extracts of high dilutions of P. amarus collected from South India inhibited HBV DNAp, DNApI, T4-DNAp, the Klenow fragment and reverse transciptase of avian myeloblastosis virus. Shead et al (1990) (1990 International symposium on viral Hepatitis and liver diseases, Apr. 4-8, 1990, Houston, Tex., USA; A602) from Australia have s the aqueous extracts to inhibit the endogenous DNAp of DHBV at high dilutions. Niu etal (1990) form Australia in collaboration with Thyagarajan from India using P. amarus collected from Madras, Tamilnadu, on of 4-5 week old ducks congenitally infected with Duck hepatitis B virus (DHBV) with suitable controls after a period of 10 weeks treatment showed transient reduction of viral DNA in serum but no effect on the level of virus DNA or surface antigen in the liver (J Med Virol 1990;32:212-218).
Jayaram et al (1996) (Ind J Pathol Microbiol 1996;39(3):211-215) reported invitro inhibition of HBsAg secretion by PLC/PRF/5 (Alexander) cellline for 48 hrs when the cellline was treated with 1 mg/ml concentration of P. amarus as a single dose. Lee et al (1996) (European J Clin Invest 1996; 26:1069-76) from USA in collaboration with Thyagarajan have shown that P. amarus down-regulates Hepatitis B virus m RNA transcription and replication using transgenic mice and transgenic celllines. The continuation of this collaboration by Ott et al (1997) has shown the cellular and molecular mechanism of HBV suppression by P. amarus to be by interrupting interactions between HBV enhancer I and cellular transcription factors (European 3 Clin Invest 1997; 27:908-915).
The biosafety studies on P. amarus dates back to 1971 when Mokkshasmit et al from Thailand using P. niruri have reported it to be non toxic to mice at 10 gms/kg body weight (Bull of Dept of Medical Science NAPRALERT Chicago, Ill., 1971;12:36-65). Rao (1985) from Andhra Pradesh, India reported 20% aqueous extct of P. niruri leaves to be effective to be as an oral pretreatment of 0.2 ml/100 mg body weight against CCl4 induced hepatotoxicity in rats (Probe 1985;115-119). Syamasundar et al (1985) from Uttar Pradesh, India showed the hexane extracted compounds Phyllanthin and Hypo phyllanthin reduced CCl4 or galatosamine induced cytotoxicity to cultured rat hepatocytes (J Ethnopharmacol 1985;14:41-44). Jayaram et al (1987) from Madras, India using the aqueous extract of dried whole plant showed no chronic toxicity in mice at 0.2 mg daily dose per animal for 90 days as revealed by physiological, biochemical and histopathological parameters. There was also no cytotonic or cytotoxic changes when tested in a tissue culture model using vero cell line (Biomedicine 1987;7:9-16). Venkateswaran et al (1987) from USA demonstrated its invivo safety using woodchucks as animal models, while Niu et al (1990) from Australia have shown P. amarus to be non toxic in Pekin ducks chronically infected with duck Hepatitis B virus. Jayaram and Thyagarajan (1994) studying the effect of P. amarus on xcex2-galactosamine induced hepatoxicity on isolated rat hepatocytes have shown that a) P. amarus by itself did not bring about any hepatotoxicity on rat hepatocytes. b) At 1 mg/ml concentration the aqueous extract were shown to protect isolated rat hepatocytes significantly from xcex2-galactosamine induced hepatoxicity thus proving the anti hepatoxicity potentials of P. amarus (Ind J Med Microbiol 1994;12(4):247-250).
In all the traditional medicine systems, there has been several formulatory medicines for the treatment of jaundice in general without taking into consideration their viral etiology. Even though P. niruri was one of the constituents of such medicines, these were always been multiherbal preparations containing anywhere upto 12 medicinal herbs and most of the treatment evaluations were based on clinical improvement only. On the other hand, there is no documented trial report on their use in chronic liver disease patients.
It was in this context Thyagarajan and his collaborators, after proving the invitro and invivo efficacy and safety of P. amarus conducted 2 open clinical trials in acute viral hepatitis cases and seven clinical trials (2 of them being double blind trials and the others Phase I/II open trials) in chronic carriers of Hepatitis B virus (HBV). Jayanthi et al (1988) ( Gastroenterol and Hepatol 1988; 3:533-534) in a control clinical trial in acute viral hepatitis (AVH) using P. niruri on one arm, and other herbal medicines in other groups have shown a significantly greater decrease in transaminases after two weeks treatment with P. niruri in both HBsAg positive and negative groups. In a virologically characterised AVH clinical trial, Geetha et al (1992) (J Gen Medicine 1992;4(2):53-58) have shown that a) P. amarus treatment has brought about significantly faster biochemical normalcy in both hepatitis A and B; b) there was a higher rate of HBsAg clearance in P. amarus treated AVH-B cases than other treatment modalities and c) there was no observable side effects due to P. amarus treatment.
The clinical trials were conducted by Thyagarajan et al between 1988 and 1997. While the first trial 1988 (Lancet 1988; 2:764-766) reported 59% HBsAg clearance in the P. amarus treated group, as against 4% in the placebo group, the second open trial (1990) (Lancet 1990;2: 949-950) showed 20% HBsAg clearance and 63.6% loss of infectivity indicated by HBeAg sero-conversion. Parallely, investigators from other countries like Leeiarasamee et al (1990) (Lancet 1990;1:1600-1601) from Thailand, Wang Me Xia et al from China (1991) (Hepatology RLR 1991;21(5):22-24) have reported the non reproducibility of treatment efficacy by the local variety of P. amarus grown in their respective countries.